Tau Seeds Research Articles

Modeling tauopathy using human-like pathogenic tau seeds.

Compared with traditional transgenic models of tauopathy, there is no confounding effect from the insertion of the exogenous gene and overexpression of the human mutant tau in the tau spreading models. By strategically choosing the location and time of the inoculation, we can spatiotemporally determine the starting point of tau pathology in the model mimicking the development of tauopathy. Moreover, they recapitulate the strain-specific features of tau pathology. Regardless of these advantages, human-like tau spreading models are very much confined due to the limitation on quantity and quality of tau seeds. In vitro seeding assay has shown its great potential of amplifying the pathogenic tau seeds to expand their quantity and increase their quality. In the study, we used biochemically enriched, human-derived, tau aggregates as seeds to template recombinant tau (rT40) in vitro. Using biochemistry and electron microscopy, we examined the biophysical properties of the amplified materials and the human-derived tau seeds. We also interrogated the pathogenicity of the amplified human-like tau strains with orthogonal spreading models of tauopathy. As a result, we observed elongated tau filaments with recombinant T40 attached to the seeds. Compared with the seeds control, the amplified tau strains showed increased pathogenic tau species after seeding. The amplified tau strains recapitulate the pathogenicity and biophysical property of the original seeds. Our data suggest that the in vitro seeded recombinant tau can be recruited onto the seeds and converted into the confirmation of pathological tau seeds in a strain-dependent manner. Along with the conversion, the pathogenicity of different tau strains can be conferred and conserved into the recombinant tau. The study provides an authentic way of amplifying tau strains in vitro as well as a tool to expand our ability to understand tau fibrillization and transmission.

Investigating astrocytes as mediators of tau spread.

Highly phosphorylated tau aggregates spread in a prion-like manner through tauopathy brain. Increasing evidence suggests that astrocytes may influence tau spread, with recent data describing mechanisms of tau uptake my astrocytes. However, the efficiency of uptake of different disease-associated tau species is not well defined, nor is the effects of tau uptake on astrocyte reactivity and function. We have investigated the relationship between tau seeds and reactive astrocytes in htau mice and in human astrocyte cultures. Tau was isolated from human postmortem control (CTRL), Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and Pick's disease (PiD) brain and injected intraperitoneally into 4-month old wild-type and htau mice. Brains and other tissues were collected for biochemical and immunohistochemical analysis of tau aggregated burden in different brain regions when mice reached 18 months of age. Human iPSC-derived astrocytes and iNPC-astrocytes were established and their gene and protein expression profiles characterised. Aggregated tau was isolated from postmortem human brain for spiking into astrocyte cultures. Cells were analysed for efficiency of tau uptake and astrocyte reactivity. Peripheral injection of brain extracts from AD, PSP and PiD brain resulted in regional differences in the patterns of tau aggregate accumulation in htau mouse brain. Tau pathology was commonly associated with reactive astrocytes. Cultures of human iPSC-derived astrocytes and iNPC-astrocytes that express mature astrocyte markers were established. Both types of cell culture were found to internalise tau extracts from human postmortem brain. Furthermore, preliminary analysis indicates alterations in astrocyte reactivity following tau uptake. Our data suggests that neurons in different regions are selectively vulnerable to tau aggregates from different tauopathies. Our human astrocyte cultures demonstrate that astrocytes can internalise tau seeds and alter their reactivity and potentially their functions. Further investigations will determine the mechanisms of tau uptake and the downstream effects on astrocyte signalling and functional changes, providing further insights into how astrocytes may affect the spread of tau and potentially contribute to selective regional vulnerability in tauopathies.

Rate‐limiting processes of tau aggregate accumulation in Alzheimer's disease

AbstractBackgroundBoth the replication of tau aggregates and their spreading throughout the brain are implicated in the progression of Alzheimer’s disease (AD). However, the rates of these processes are unknown and the identity of the rate‐determining process in humans has therefore remained elusive. Using a mathematical framework based on chemical kinetics, we are able to answer these questions by analysis of experimental data from AD patients.MethodOur minimal mathematical model of aggregate spreading and replication enables us to determine the rates of spreading and replication from measurements of tau seeds and aggregates across brain regions. Simulations based on this model show clearly distinct patterns of tau distributions when the behaviour is limited by either replication or spreading (Fig 1). We analysed several already existing datasets from in vitro seeding experiments, tau pathology in P301S transgenic mice, and histochemical post‐mortem as well as 2‐year longitudinal tau PET data in humans (Fig 2).ResultAcross mice and human data, we found that the overall rate of accumulation of tau in cortical regions is limited not by the rate of tau spreading between brain regions but by the rate of local replication within a brain region. In human post‐mortem data, low levels of tau seeds were present already throughout the cortex at Braak stage 3, where effective replication of those initial seeds rather than inter‐regional spreading was the rate limiting factor for further tau accumulation. The rate of replication determined from these data corresponds to a doubling of the number of seeds only every ∼5 years, which was well in line with the our analysis of tau accumulation from longitudinal tau PET data in AD patients. This rate is orders of magnitude slower than that measured for purified tau in vitro, quantifying the efficiency of innate cellular mechanisms curtailing tau seed replication.ConclusionBased on these findings, we believe that limiting local replication constitutes the most promising strategy to control tau accumulation during AD, after Braak stage 2. Our results provide a framework for the determination of the identity and the rates of the key steps controlling disease‐associated aggregation processes within living organisms.

Quantifying acute tau inoculations in living rats using PET

AbstractBackgroundMonitoring tau propagation in the living brain has important pathophysiological and therapeutic implications. As tau imaging agents are dependent on tau conformations, we aimed to determine the tau species amenable to in vivo imaging. Previously we demonstrated that ex vivo tau preparations may be used to image tau seeds in living murine systems, as opposed to recombinant preparations. As such, the goal of this work is to examine the dose‐dependent relationship of patient derived AD‐tau fibrils on PET imaging outcomes.MethodA total of two 15‐month‐old (2 wild‐type, one male and one female) rats were used. All animal had a baseline scan with [18F] MK6240 before the stereotaxic injections. We tested tau PHFs from AD brains using ultracentrifugation of sarkosyl‐insoluble brain fractions. AD‐tau preparations were stereotaxically injected in the dorsal hippocampus. We tested injection volumes of 1 and 3 uL of equal concentrations (80 ng/uL). Contralateral hemispheres were injected with equivalent amounts of PBS, the solvent for tau preparations. All PET images were coregistered to the Waxholm Space Atlas of the Sprague Dawley Rat Brain sMRI using a linear transformation. The non‐displaceable binding potential (BPND) images were generated using cerebellar grey as a reference region based on Simplified Reference Tissue Method (SRTM).ResultInjection of sarkosyl‐insoluble brain fractions from AD patients displayed positive BPnd outcome in both volumes tested. Control hemispheres injected with PBS showed potential off‐target binding. Reliability of this outcome was investigated using peak normalized TACs. Hemispheres receiving AD‐tau injections showed visible divergence in retention, as compared to baseline. The hemisphere that was inoculated with 3uL of AD‐tau showed separation during washout. A similar effect was seen in the control hemisphere.ConclusionThese results continue to suggest ex vivo tau preparations may be used to image tau seeds in living murine systems. The reliability of PET and kinetic outcomes are dependent on the volume injected. Further investigation will aim to evaluate AD‐tau concentration as the dosage variable instead of injection volume. Cerebellar tissue from patients used to extract AD‐tau species should be as a control injection to account for the reagents used for protein extraction, instead of PBS alone.

Targeting aggregated tau turnover as a therapeutic strategy

The accumulation of microtubule-associated protein tau in intracellular inclusions is a common feature of Alzheimer's disease and other tauopathies. We have recently identified using optical pulse labelling methodology (OPL) that neurofibrillary tau inclusions comprised of aggregated tau are dynamic structures that show turnover of aggregated tau. This occurs in both intrinsically formed and exogenously seeded tau inclusions. Understanding the mechanisms by which this turnover occurs could inform how to most appropriately treat tauopathies.Using recombinant adeno-associated viruses (rAAVs) we are able to deliver Dendra2-tagged pro-aggregant and non-aggregating tau to murine brain slice cultures with and without the application of exogenous tau seeds. These methods facilitate the long-term tracking of tau inclusion dynamics using OPL methodology. In combination with these methods, we can begin to assess whether compounds, monoclonal antibodies or other potential therapeutic modulators change tau aggregate turnover or affect tau by differential mechanisms.Using OPL, we can track the formation of neurofibrillary tau inclusions and the kinetics of tau turnover. We find that newly formed tau inclusions show turnover, but with ageing in culture the rate of turnover slows. Using this experimental paradigm, we now aim to begin to understand the mechanisms that facilitate this turnover and determine whether therapies that affect this turnover may be beneficial. We also find that a compound known to reduce tau pathology reduces tau phosphorylation and tau insolubility but does not affect the rate of tau production or clearance.Our OPL studies have documented that neurofibrillary tau inclusions comprised of aggregated tau show daily turnover of tau, at least when initially formed. We now aim to use this system to begin to understand the cellular processes that facilitate this tau inclusion turnover. Furthermore, understanding these mechanisms may lead to new therapeutic strategies for Alzheimer's disease and other neurodegenerative conditions where tau plays a role.

P62 accumulates through neuroanatomical circuits in response to tauopathy propagation

In Alzheimer’s disease and related tauopathies, trans-synaptic transfer and accumulation of pathological tau from donor to recipient neurons is thought to contribute to disease progression, but the underlying mechanisms are poorly understood. Using complementary in vivo and in vitro models, we examined the relationship between these two processes and neuronal clearance. Accumulation of p62 (a marker of defective protein clearance) correlated with pathological tau accumulation in two mouse models of tauopathy spread; Entorhinal Cortex-tau (EC-Tau) mice where tau pathology progresses in time from EC to other brain regions, and PS19 mice injected with tau seeds. In both models and in several brain regions, p62 colocalized with human tau in a pathological conformation (MC1 antibody). In EC-Tau mice, p62 accumulated before overt tau pathology had developed and was associated with the presence of aggregation-competent tau seeds identified using a FRET-based assay. Furthermore, p62 accumulated in the cytoplasm of neurons in the dentate gyrus of EC-Tau mice prior to the appearance of MC1 positive tauopathy. However, MC1 positive tau was shown to be present at the synapse and to colocalize with p62 as shown by immuno electron microscopy. In vitro, p62 colocalized with tau inclusions in two primary cortical neuron models of tau pathology. In a three-chamber microfluidic device containing neurons overexpressing fluorescent tau, seeding of tau in the donor chamber led to tau pathology spread and p62 accumulation in both the donor and the recipient chamber. Overall, these data are in accordance with the hypothesis that the accumulation and trans-synaptic spread of pathological tau disrupts clearance mechanisms, preceding the appearance of obvious tau aggregation. A vicious cycle of tau accumulation and clearance deficit would be expected to feed-forward and exacerbate disease progression across neuronal circuits in human tauopathies.

Microglia become hypofunctional and release metalloproteases and tau seeds when phagocytosing live neurons with P301S tau aggregates.

The microtubule-associated protein tau aggregates in multiple neurodegenerative diseases, causing inflammation and changing the inflammatory signature of microglia by unknown mechanisms. We have shown that microglia phagocytose live neurons containing tau aggregates cultured from P301S tau mice due to neuronal tau aggregate-induced exposure of the “eat me” signal phosphatidylserine. Here, we show that after phagocytosing tau aggregate-bearing neurons, microglia become hypophagocytic while releasing seed-competent insoluble tau aggregates. These microglia express a senescence-like phenotype, demonstrated by acidic β-galactosidase activity, secretion of paracrine senescence-associated cytokines, and maturation of matrix remodeling enzymes, results that are corroborated in P301S mouse brains and ex vivo brain slices. In particular, the nuclear factor κB–dependent activation of matrix metalloprotease 3 (MMP3/stromelysin1) was replicated in brains from patients with tauopathy. These data show that microglia that have been activated to ingest live tau aggregates-bearing neurons behave hormetically, becoming hypofunctional while acting as vectors of tau aggregate spreading.

Tau seeding in chronic traumatic encephalopathy parallels disease severity.

Chronic traumatic encephalopathy (CTE), a neurodegenerative tauopathy, is associated with behavioral, mood and cognitive impairment, including dementia. Tauopathies are neurodegenerative diseases whose neuropathological phenotypes are characterized by distinct histopathologic features of tau pathology, which progressively deposit throughout the brain. In certain tauopathies, especially Alzheimer's disease (AD), tau deposition appears to follow brain network connections. Experimental evidence suggests that the progression of tau pathology in humans, mouse and cell models could be explained by tau seeds that adopt distinct conformations and serve as templates for their own amplification to mediate transcellular propagation of pathology. Tau seeds are efficiently detected by the induction of aggregation in cell-based "biosensors" that express tau repeat domain (RD) with a disease-associated mutation (P301S) fused to complementary fluorescent protein tags (cyan and yellow fluorescent protein). Biosensors enable quantification of tau seeding in fixed and fresh-frozen brain tissue. Phospho-tau deposition in CTE follows progressive stages (I-IV), but the relationship of seeding to this deposition is unclear. We have used an established biosensor assay to independently quantify tau seeding as compared to AT8 phospho-tau histopathology in thin sections of fixed tissues of 11 brain regions from 27 patients with CTE, 5 with other tauopathies, and 5 negative controls. In contrast to prior studies of AD, we detected tau seeding late in the course of CTE (predominantly stages III and IV). It was less anatomically prevalent than AT8-positive inclusions, which were relatively widespread. We especially observed seeding in the limbic system (amygdala, thalamus, basal ganglia), which may explain the dominant cognitive and behavior impairments that characterize CTE.

Impact of Sterilization Methods on the Seeding Ability of Human Tau Proteopathic Seeds.

The protein tau, when misfolded in neurodegenerative diseases, has several prion-like properties including being able to spread by cell-to-cell transfer, induce templated seeding, and exist in distinct conformational strains. These properties of transmission may present health hazards when lesion-containing biospecimens are used in research and neuropathology laboratories. We evaluated the impact standard sterilization and cleaning methods have on the capacity of tau seeds to induce aggregation. We employed a previously developed, highly sensitive FRET-based biosensor assay to assess remnant tau seeding after exposure to these procedures. For tau species derived from human Alzheimer disease tissue (brain homogenate and sarkosyl-insoluble fibrils), both autoclaving and incubation in 90.6% formic acid were sufficient to reduce tau bioactivity. By contrast, boiling was not always effective in completely blocking bioactivity in the seeding assay. Notably, only formic acid incubation was able to produce a similar reduction in tissue from a P301L mutant tau mouse model of tauopathy. Our study highlights nuances in methods for inactivation of tau seeding which may support adapted tissue processing procedures, especially in research settings. These findings also highlight the importance of universal precautions when handling human neuropathological and research laboratory materials.

Spreading of Alzheimer tau seeds is enhanced by aging and template matching with limited impact of amyloid-β

In Alzheimer's disease (AD), deposition of pathological tau and amyloid-β (Aβ) drive synaptic loss and cognitive decline. The injection of misfolded tau aggregates extracted from human AD brains drives templated spreading of tau pathology within WT mouse brain. Here, we assessed the impact of Aβ copathology, of deleting loci known to modify AD risk (Ptk2b, Grn, and Tmem106b) and of pharmacological intervention with an Fyn kinase inhibitor on tau spreading after injection of AD tau extracts. The density and spreading of tau inclusions triggered by human tau seed were unaltered in the hippocampus and cortex of APPswe/PSEN1ΔE9 transgenic and AppNL-F/NL-F knock-in mice. In mice with human tau sequence replacing mouse tau, template matching enhanced neuritic tau burden. Human AD brain tau-enriched preparations contained aggregated Aβ, and the Aβ coinjection caused a redistribution of Aβ aggregates in mutant AD model mice. The injection-induced Aβ phenotype was spatially distinct from tau accumulation and could be ameliorated by depleting Aβ from tau extracts. These data suggest that Aβ and tau pathologies propagate by largely independent mechanisms after their initial formation. Altering the activity of the Fyn and Pyk2 (Ptk2b) kinases involved in Aβ-oligomer–induced signaling, or deleting expression of the progranulin and TMEM106B lysosomal proteins, did not alter the somatic tau inclusion burden or spreading. However, mouse aging had a prominent effect to increase the accumulation of neuritic tau after injection of human AD tau seeds into WT mice. These studies refine our knowledge of factors capable of modulating tau spreading.

Tau Seeds in Extracellular Vesicles Induce Tau Accumulation in Degradative Organelles of Cells.

Clinical progression of tauopathies may result from transcellular propagation of pathogenic Tau seeds with the possible involvement of extracellular vesicles (EVs) as transport vectors. We established a cell model for investigating EV delivery of proteins, since the mechanism regulating EV cargo delivery to recipient cells is poorly understood. In our cell model, EVs are readily internalized and accumulate in degradative organelles (DOs). We then show for the first time that in this acidic compartment, profibrillogenic Tau delivered by EVs interacts with Tau expressed by the recipient cells and cause its accumulation by a process that involves the participation of autophagy. Thus, the degradative compartment of cells may represent the subcellular site initiating a cascade of events resulting in early hallmarks of tauopathies. These are characterized by seeded Tau accumulation, pathology-associated epitopes, DO stress, and cytotoxicity. The involvement of autophagy to this process and the relative accessibility of the degradative pathway for extracellular agents, support possible modes of intervention to slow down the progression of neurodegeneration.

Hsp40s play complementary roles in the prevention of tau amyloid formation.

The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles associated with neurodegenerative conditions, such as Alzheimer's disease. In the cell, however, tau aggregation can be prevented by a class of proteins known as molecular chaperones. While numerous chaperones are known to interact with tau, though, little is known regarding the mechanisms by which these prevent tau aggregation. Here, we describe the effects of ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid-fiber formation and compare these to the small heat shock protein HSPB1. We find that the chaperones play complementary roles, with each preventing tau aggregation differently and interacting with distinct sets of tau species. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone conformers and even mature fibers. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, distinct site in CTDI. These results lay out the mechanisms by which the diverse members of the Hsp40 family counteract the formation and propagation of toxic tau aggregates and highlight the fact that chaperones from different families/classes play distinct, yet complementary roles in preventing pathological protein aggregation.

Biophysical properties of a tau seed

Pathogenesis of tauopathies involves conversion of tau monomer into pathological tau conformers that serve as templates to recruit native tau into growing assemblies. Small soluble tau seeds have been proposed to drive pathological tau assembly in vitro, in cells and in vivo. We have previously described the isolation of monomeric pathogenic tau seeds derived from recombinant samples and tauopathy tissues but in-depth biophysical characterization of these species has not been done. Here we describe a chromatographic method to isolate recombinant soluble tau seeds derived from heparin treatment. We used biochemical and biophysical approaches to show that the seeds are predominantly monomeric and have the capacity to nucleate aggregation of inert forms of tau in vitro and in cells. Finally, we used crosslinking mass spectrometry to identify the topological changes in tau as it converts from an inert state to a pathogenic seed. Future studies will reveal the relationship between soluble seeds and structural polymorphs derived from tauopathies to help diagnose and develop therapeutics targeting specific tauopathies.

Exosomal and vesicle-free tau seeds-propagation and convergence in endolysosomal permeabilization.

In Alzheimer's disease (AD), β-amyloid peptides aggregate to form amyloid plaques, and the microtubule-associated protein tau forms neurofibrillary tangles. However, severity and duration of AD correlate with the stereotypical emergence of tau tangles throughout the brain, suggestive of a gradual region-to-region spreading of pathological tau. The current notion in the field is that misfolded tau seeds propagate transsynaptically and corrupt the proper folding of soluble tau in recipient neurons. This is supported by accumulating evidence showing that in AD, functional connectivity and not proximity predicts the spreading of tau pathology. Tau seeds can be found in two flavors, vesicle-free, that is, naked as in oligomers and fibrils, or encapsulated by membranes of secreted vesicles known as exosomes. Both types of seeds have been shown to propagate between interconnected neurons. Here, we describe potential ways of how their propagation can be controlled in several subcellular compartments by manipulating mechanisms affecting production, neuron-to-neuron transmission, internalization, endosomal escape, and autophagy. We emphasize that although vesicle-free tau seeds and exosomes differ, they share the ability to trigger endolysosomal permeabilization. Such a mechanistic convergence in endolysosomal permeabilization presents itself as a unique opportunity to target both types of tau seeding. We discuss the cellular response to endolysosomal damage that might be key to control permeabilization, and the significant overlap in the seeding mechanism of proteopathic agents other than tau, which suggests that targeting the endolysosomal pathway could pave the way toward developing broad-spectrum treatments for neurodegenerative diseases.

Ultrasensitive tau biosensor cells detect no seeding in Alzheimer\u2019s disease CSF

Tau protein forms self-replicating assemblies (seeds) that may underlie progression of pathology in Alzheimer’s disease (AD) and related tauopathies. Seeding in recombinant protein preparations and brain homogenates has been quantified with “biosensor” cell lines that express tau with a disease-associated mutation (P301S) fused to complementary fluorescent proteins. Quantification of induced aggregation in cells that score positive by fluorescence resonance energy transfer (FRET) is accomplished by cell imaging or flow cytometry. Several groups have reported seeding activity in antemortem cerebrospinal fluid (CSF) using various methods, but these findings are not yet widely replicated. To address this question, we created two improved FRET-based biosensor cell lines based on tau expression, termed version 2 low (v2L) and version 2 high (v2H). We determined that v2H cells are ~ 100-fold more sensitive to AD-derived tau seeds than our original lines, and coupled with immunoprecipitation reliably detect seeding from samples containing as little as 100 attomoles of recombinant tau fibrils or ~ 32 pg of total protein from AD brain homogenate. We tested antemortem CSF from 11 subjects with a clinical diagnosis of AD, 9 confirmed by validated CSF biomarkers. We used immunoprecipitation coupled with seed detection in v2H cells and detected no tau seeding in any sample. Thus we cannot confirm prior reports of tau seeding activity in the CSF of AD patients. This next generation of ultra-sensitive tau biosensors may nonetheless be useful to the research community to quantify tau pathology as sensitively and specifically as possible.

The Sulfation Code of Tauopathies: Heparan Sulfate Proteoglycans in the Prion Like Spread of Tau Pathology.

Tauopathies are a heterogenous family of progressive neurodegenerative diseases defined by the appearance of proteinaceous lesions within the brain composed of abnormally folded species of Microtubule Associated Protein Tau (tau). Alzheimer’s Disease (AD), the most common tauopathy, is the leading cause of cognitive decline among the elderly and is responsible for more than half of all cases of senile dementia worldwide. The characteristic pathology of many tauopathies—AD included—presents as Neurofibrillary Tangles (NFTs), insoluble inclusions found within the neurons of the central nervous system composed primarily of tau protein arranged into Paired Helical Fibrils (PHFs). The spatial extent of this pathology evolves in a remarkably consistent pattern over the course of disease progression. Among the leading hypotheses which seek to explain the stereotypical progression of tauopathies is the prion model, which proposes that the spread of tau pathology is mediated by the transmission of self-propagating tau conformers between cells in a fashion analogous to the mechanism of communicable prion diseases. Protein-glycan interactions between tau and Heparan Sulfate Proteoglycans (HSPGs) have been implicated as a key facilitator in each stage of the prion-like propagation of tau pathology, from the initial secretion of intracellular tau protein into the extracellular matrix, to the uptake of pathogenic tau seeds by cells, and the self-assembly of tau into higher order aggregates. In this review we outline the biochemical basis of the tau-HS interaction and discuss our current understanding of the mechanisms by which these interactions contribute to the propagation of tau pathology in tauopathies, with a particular focus on AD.

Continuous Monitoring of Tau-Induced Neurotoxicity in Patient-Derived iPSC-Neurons.

Tau aggregation within neurons is a critical feature of Alzheimer's disease (AD) and related tauopathies. It is believed that soluble pathologic tau species seed the formation of tau aggregates in a prion-like manner and propagate through connected neurons during the progression of disease. Both soluble and aggregated forms of tau are thought to have neurotoxic properties. In addition, different strains of misfolded tau may cause differential neurotoxicity. In this work, we present an accelerated human neuronal model of tau-induced neurotoxicity that incorporates both soluble tau species and tau aggregation. Using patient-derived induced pluripotent stem cell (iPSC) neurons expressing a tau aggregation biosensor, we develop a cell culture system that allows continuous assessment of both induced tau aggregation and neuronal viability at single-cell resolution for periods of >1 week. We show that exogenous tau “seed” uptake, as measured by tau repeat domain (TauRD) reporter aggregation, increases the risk for subsequent neuronal death in vitro. These results are the first to directly visualize neuronal TauRD aggregation and subsequent cell death in single human iPSC neurons. Specific morphologic strains or patterns of TauRD aggregation are then identified and associated with differing neurotoxicity. Furthermore, we demonstrate that familial AD iPSC neurons expressing the PSEN1 L435F mutation exhibit accelerated TauRD aggregation kinetics and a tau strain propagation bias when compared with control iPSC neurons.SIGNIFICANCE STATEMENT Neuronal intracellular aggregation of the microtubule binding protein tau occurs in Alzheimer's disease and related neurodegenerative tauopathies. Tau aggregates are believed to spread from neuron to neuron via prion-like misfolded tau seeds. Our work develops a human neuronal live-imaging system to visualize seeded tau aggregation and tau-induced neurotoxicity within single neurons. Using an aggregation-sensing tau reporter, we find that neuronal uptake and propagation of tau seeds reduces subsequent survival. In addition, human induced pluripotent stem cell (iPSC) neurons carrying an Alzheimer's disease-causing mutation in presenilin-1 undergo tau seeding more rapidly than control iPSC neurons. However, they do not show subsequent differences in neuronal survival. Finally, specific morphologies of tau aggregates are associated with increased neurotoxicity.

Nasal Extracts from Patients with Alzheimer's Disease Induce Tau Aggregates in a Cellular Model of Tau Propagation.

Background:Emerging evidence indicates that the misfolded tau protein can propagate aggregates between cells in a prion-like manner. This prion activity has been typically studied in brain extracts of patients with Alzheimer’s disease (AD), but not in the olfactory region that can be a potential biomarker in AD.Objective:To investigate the prion seeding activity of tau in nasal mucosa tissues using a cell culture model of tau propagation.Methods:Brain and nasal mucosa homogenates were added to HEK293T cells expressing three repeat or four-repeat domains of tau with the L266V, V337M (3RD*VM) and P301L and V377M mutations (4RD*LM) fused to the enhanced green fluorescence protein (EGFP) respectively. We also measured the level of phosphorylated tau (p-tau), total tau (t-tau), and p-tau/t-tau ratio and performed correlation analysis between tau prion activity and the level of tau.Results:We found that brain and nasal tissue homogenates from patients with AD significantly induced tau aggregation in HEK293T cells either expressing tau 3RD*VM-EGFP or 4RD*LM-EGFP compared with control brain and nasal tissue homogenates. The levels of p-tau and p-tau/t-tau ratio were significantly increased in the brain of patients with AD; however, no significant difference was found in nasal tissue compared with their respective control tissue homogenates.Conclusion:These results suggest that the nasal tissues contain tau seeds, similar to the brain, albeit without changes in the levels of p-tau and t-tau. Therefore, a cellular bioassay using nasal tissues would have great potential as an AD biomarker because of the usefulness of nasal tissue biopsy.

Tau seeds are subject to aberrant modifications resulting in distinct signatures.

The prion-like spread of tau pathology could underlie a spectrum of clinical syndromes including Alzheimer's disease (AD). Although evidence indicates that tau is transmissible, it is unclear how pathogenic tau seeds are processed in neurons. Here, we analyze fibrillar wild-type and disease-associated P301L tau seeds by using invitro and neuronal assays. We show that P301L seeds are uniquely modified by post-translational modifications (PTMs) within the microtubule-binding region (MTBR). Although these modifications do not alter tau seed trafficking or localization, acetylated tau variants show accelerated tau aggregation, enhanced tau PTM priming, and prion-like templating. To explain the enhanced tau seed acetylation, we demonstrate that P301L seeds undergo auto-acetylation. Moreover, tau acts generally to inhibit HDAC6 deacetylase activity by preventing HDAC6 phosphorylation, leading to increased substrate acetylation. Our study highlights complex post-translational regulation of transmissible tau seeds and provides insight into the biological properties of tau strains in AD and other tauopathies.

Quantitative Methods for the Detection of Tau Seeding Activity in Human Biofluids.

The ability of tau aggregates to recruit and misfold monomeric tau and propagate across brain regions has been studied extensively and is now recognized as a critical pathological step in Alzheimer’s disease (AD) and other tauopathies. Recent evidence suggests that the detection of tau seeds in human samples may be relevant and correlate with clinical data. Here, we review the available methods for the measurement of such tau seeds, their limitations and their potential implementation for the development of the next-generation biomarkers.